1. Field of the Invention
The present invention relates generally to optical scanning devices, and more particularly, to multi-photon fluorescence microscopes.
2. Related Art
Optical scanning devices, e.g. laser scanning microscopes are used to scan samples multi-dimensionally by directing a beam of light over the sample's surface to obtain a multi- (two- or three-) dimensional view.
If fast effects, e.g. structural re-arrangements or chemical reactions, are to be measured, the scanning must be correspondingly fast. A faster scanning can be achieved by moving the beam faster across the surface.
In most cases one cannot compensate for that by simply increasing the illumination intensity since this increasingly leads to saturation of the fluorescence dye, to stronger bleaching or even worse to damaging processes in the sample. This is also true for multi-photon fluorescence microscopes. Here the fluorescent molecules, e.g. GFP, of an object/sample are excited by the high intensity of a short pulse laser so that two photons (typically in the near infrared) are absorbed simultaneously to reach the excitation band of the fluorescence molecule in the green or blue range. A specific advantage of the multi-photon fluorescence excitation is that it is less photo-toxic and has larger penetration depth due to the longer wavelength of the red light, as opposed to short-wave/blue light. Unfortunately the so-called two-photon effect is a rather weak interaction that produces relatively few fluorescence photons compared to the single photon excitation. Thus, in order to get a reasonable signal-to-noise ratio, a longer measurement period or higher excitation intensity would be needed. One way to limit or shorten this measurement period without a damaging increase in the light intensity is to use multiple beams simultaneously with intensities below the detrimental thresholds. The generation of multiple secondary beams (beamlets) out of the primary beam can be done by arrays of micro-lenses. These beamlets then simultanously scan different parts of the object, thus reducing overall measurement time roughly by the number of beamlets. An array of micro-lenses is described in A. Egner, S. Jacobs, S. W. Hell: “Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast” pp. 3370-3375, PNAS, 19 Mar. 2002, vol. 99 no. 6, or: A. Egner et al.: Time multiplexing and parallelization in multifical multiphoton microscopy”, J. Opt. Soc. Am. A/Vol. 17, No. 7/July 2000.
Another way to split the primary beam into multiple beamlets is to use multiple beam splitters and mirrors, as e.g. described in DE 199 04 592 C2.
With both above mentioned beam splitting methods it is difficult to get beamlets of equal intensity and quality, particularly for several wave lengths. For example, the arrays of micro-lenses show significant aberration errors and production tolerances, esp. with respect to the focus lengths. Additionally, the motorisation required to move the lenses causes mechanical vibration effects in the instrument disturbing the microscopic imaging, especially at high resolutions.
FR 2 814 247 shows a confocal laser microscope that can be especially applied to fluorescent measurements in biological samples. To speed up the measurement process, the collimated laser-beam can by divided into multiple beams of different directions by means of birefringent prisms.
In a fundamentally different microscope technique a non focussed wide field beam is split up into two slightly inclined beams by a Wollaston prism in order to generate the so-called Nomarski Interference Contrast or Differential Interference Contrast (DIC). This technique allows to turn a microscopic phase object optically into an amplitude object, so that a visual contrast is generated. Here the two beams are displaced in the focus by a fraction of the optical resolution with respect to each other. With displacements larger than the optical resolution the interference and thus the contrast vanishes.
US 2001/0012151 A1 shows a microscope assemblage, in particular for confocal scanning microscopy, having a light source for illuminating a specimen to be examined and at least one fluorescent-light detector for the detection of fluorescent light generated in the specimen and at least one transmitted-light detector for the detection of transmitted light passing through the specimen. The microscope is configured such that the fluorescent-light and transmitted-light detected are arranged in such a way as to make possible simultaneous detection of fluorescent and transmitted light. This microscope can be used in particular for interference contrast microscope. For this purpose a first polarisation device could be arranged between the light source and the specimen, preferably before the objective, and a second polarisation device after the specimen, preferably after the condenser. The polarisation devices could be constituted by prisms, wherein in this context Wollaston prisms are particularly suitable.
U.S. Pat. No. 6,392,752 B1 shows a device for phase-measuring microlens microscopy, wherein a scanning microlens array functions in a manner analogous to an array of interference microscopes to provide phase-sensitive, confocal micro-imaging capability. Moreover, the scanning mechanism can effectively perform a phase-modulation function. In this mode of operation, each image point is scanned by multiple microlenses that have fixed, but different, built-in phase offsets, and the combination of signal acquired from the multiple scans effectively simulate a phase-modulated interference signal. The scanning confocal microlens array can be adapted to provide phase-measuring capability by equipping each microlens with a beam-splitting mechanism that separates the illumination on the microlens into two beams, at least one of which is focused onto and reflect off an inspection sample. The two beams are coherently recombined by the beam-splitting mechanism and are projected onto an element of a detector array, wherein each detector element senses radiation from a particular corresponding microlens. The sample is scanned laterally across the focal point array to build up a synthesised, two-dimensional image of the sample surface.
Prisms, including the Wollaston prism are, e.g., described in Naumann/Schröder: “Bauelemente der optik”, 6th ed., pp. 504-509.
It is an object of the present invention to provide an optical device and a respective method that show improved beamlet characteristics and thus fast imaging at high fidelity and easier interpretation of data.
The object is solved by an optical scanning device with the features of claim 1. Advantageous scanning devices are given in the subclaims.